Sigma receptors are saturable, high affinity binding sites for several important classes of psychotropic drugs, including typical antipsychotic, antidepressant, anticonvulsant, and psychotomimetic compounds. They are likely to contribute to the beneficial and/or side-effect profile of these compounds. Sigma sites are present not only in the central nervous system but also occur in several peripheral tissues, and are expressed in high density in a number of tumor cell lines. Furthermore, they are more highly expressed in rapidly proliferating cells than in quiescent cells. They may thus play an important role in cell function. Our studies have shown that agonists at sigma-2 receptors induce morphological changes, followed by apoptotic cell death. These effects were found to occur in various tumor cell lines, as well as in primary cultures of neuronal cells. Sigma-2 agonists induce both a transient and a sustained rise in intracellular calcium levels in human SK-N-SH neuroblastoma cells, which may play a role in the induction of apoptosis. The calcium is released from intracellular stores, and is apparently independent of IP3. Furthermore, sigma-2 receptor-induced apoptosis in breast tumor cell lines was found to occur by a novel pathway which was both p53- and caspase-independent. In this period, possible involvement of the sphingolipid pathway was investigated. Sphingolipids have been shown to play an important role in cell proliferation. The sphingolipid, ceramide, can produce variable effects on cells ranging from increased cell proliferation, mediated by ceramide-activated protein kinase (CAPK), to apoptosis, mediated by ceramide-activated protein phosphatase (CAPP). Like sigma-2 agonists, ceramide (in certain cell lines) induces apoptosis by a caspase-independent mechanism. In addition, sphingoid bases such as sphingosine-1-phosphate and sphingosylphosphorylcholine (SPC) can release intracellular calcium by IP3-independent mechanisms. Sigma-2 receptor agonists were found to increase the level of ceramide in metabolically labeled neuroblastoma and breast tumor cell lines. This was usually accompanied by a decrease in sphingomyelin levels. Both effects were blocked by the novel sigma-2 antagonist, AC927, which we developed in the last reporting period. Since sigma-2 agonists did not increase sphingomyelinase activity, the usual source of ceramide in apoptotic signaling, sigma-2 receptors cause an increase in ceramide by some other mechanism. We thus investigated sphingolipid-ceramide N-deacylase (SCDase) activity. This enzyme acylates sphingosine to form ceramide and deacylates sphingomyelin to form SPC. Sigma-2 receptor agonists were found to increase formation of SPC from labeled sphingomyelin and also to increase formation of ceramide when labeled palmitic acid was used. This demonstrated both the deacylation and acylation functions. Thus, sigma-2 receptors may acutely activate SCDase to increase SPC, which transiently releases calcium from the endoplasmic reticulum. More chronic activation of SCDase by sigma-2 receptors may result in ceramide accumulation, which induces apoptosis. Further study of this pathway is currently underway. In this period, we have also begun studies aimed at cloning the sigma-2 receptor gene and isolation of the protein. Using human MCF-7 cells, a PCR product of ~600 bp with the correct molecular size as the sigma-2 receptor was found using primers directed for the human sigma-1 receptor (which is ~700 bp). This cDNA product was excised, cut with Eco R1, and ligated into pCMV-5 vector. Cloning and sequencing resulted in a 579 bp cDNA which predicted a protein identical to the sigma-1 receptor, except for a 31 amino acid deletion which started at the boundary between exon 2 and exon 3. This is downstream of the putative transmembrane region of the sigma-1 receptor. This product could be a splice variant of the sigma-1 receptor or could represent the sigma-2 receptor. RT-PCR showed that this sequence is distributed in several tissues, including brain, liver, kidney, heart, and intestine. The cDNA was expressed in COS-1 cells. Preliminary ligand binding data showed no increase in sigma-1 receptor binding, but a 4-fold increase in sigma-2-like binding activity. Current studies are aimed at improving expression of the protein in order to do a full pharmacological characterization of the receptor profile to verify identity as the sigma-2 receptor.